Method for the synthesis, compositions and use of 2&#39;-deoxy-5-fluoro-3&#39;-thiacytidine and related compounds

ABSTRACT

The present invention relates to a method of preparing the antiviral compounds 2&#39;-deoxy-5-fluoro-3&#39;thiacytidine (FTC) and various prodrug analogues of FTC from inexpensive precursors with the option of introducing functionality as needed; methods of using these compounds, particularly in the prevention and treatment of AIDS; and the compounds themselves. This synthetic route allows the stereoselective preparation of the biologically active isomer of these compounds and related compounds.

ACKNOWLEDGEMENT

The invention described herein was made with Government support undergrants no. AI-28731 and no. AI-26055 awarded by the National Institutesof Health. The Government has certain rights in this invention.

REFERENCE TO CO-PENDING APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 07/473,318,filed on Feb. 1, 1990, the contents of which are incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention relates to the use of and methods and compositionsfor preparing antiviral nucleoside analogues, particularly FTC(2'-deoxy-5-fluoro-3'-thiacytidine) and prodrug analogues of FTC. Moreparticularly, the invention relates to the B-isomers of these compoundsand their selective synthesis and use as antiviral agents.

In 1981, documentation began on the disease that became known asAcquired Immune Deficiency Syndrome (AIDS), as well as its forerunnerAIDS Related Complex (ARC). Since that time, the World HealthOrganization (WHO) has confirmed that 300,000 people have been reportedto have developed AIDS. Of these, over 150,000 are in the United States.

In 1983, the cause of the disease AIDS was established as a virus namedthe human immunodeficiency virus type 1 (HIV-1). As of December, 1990,the WHO estimates that the number of people who are infected with thevirus is between 8 and 10 million worldwide and of that number, between1,000,000 and 1,400,000 are in the U.S. Usually, a person infected withthe virus will eventually develop AIDS; in all known cases of AIDS thefinal outcome has always been death.

The disease AIDS is the end result of HIV infection. The virionreplication cycle begins with the virion attaching itself to the hosthuman T-4 lymphocyte immune cell through the bonding of a receptor onthe surface of the virion's protective coat (gp 120) with a glycoproteinon the lymphocyte cell (CD4). Once attached, the virion fuses with thecell membrane, penetrates into the host cell, and uncoats its RNA. Thevirion enzyme, reverse transcriptase, directs the process oftranscribing the RNA into single stranded DNA. The viral RNA is degradedand a second DNA strand is created. The now double-stranded DNA isintegrated into the T-cell genome.

The host cell uses its own RNA polymerase to transcribe the integratedDNA into viral RNA and the viral RNA directs the production ofglycoproteins, structural proteins and viral enzymes for the new virion,which assemble with the viral RNA intact. Once all the components areassembled, the virus buds out of the cell. Thus, the number of HIV-1virions grows while the number of T-4 lymphocytes declines.

There are at least three critical points in the virion's replicationcycle which have been identified as targets for antiviral drugs: (1) theinitial attachment of the virion to the T-4 lymphocyte (CD4glycoprotein), (2) the transcription of viral RNA to viral DNA, and (3)the assemblage of the new virions during replication.

It is the inhibition of the virus at the second critical point, theviral RNA to viral DNA transcription process, that has provided the bulkof the therapies used in treating AIDS. This transcription must occurfor the virion to replicate because the virion's genes are encoded inRNA. By introducing drugs that block the enzyme, reverse transcriptase,from transcribing viral RNA to viral DNA successfully, HIV-1 replicationcan be stopped.

After phosphorylation, nucleoside analogues, such as3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine (DDC),2',3'-didehydro-3'-deoxythymidine (D4T), 2',3'-dideoxyinosine (DDI), andvarious 2'-fluoro-derivatives of these nucleosides are relativelyeffective in halting HIV replication by inhibiting reversetranscription. Another promising anti-AIDS drug is2'-deoxy-3'-thiacytidine (BCH-189), which contains an oxathiolane ringinstead of the sugar moiety in the nucleoside. This invention providesthe new antiviral nucleosides, 2'-deoxy-5-fluoro-3'-thiacytidine (FTC)and various prodrug analogues of FTC, which are unexpectedly potent andnontoxic.

AZT is a successful anti-HIV drug because it prevents the nucleotidechain-linking reaction that elongates viral DNA inside the host T-4lymphocyte cells or other immune system cells such as macrophages. WhenAZT enters the cell, cellular kinases activate AZT by phosphorylation toAZT triphosphate. AZT triphosphate then competes with natural thymidinenucleotides for the receptor site of HIV reverse transcriptase enzyme.The natural nucleotide possesses two reactive ends, the 5'-triphosphateend which reacts with the growing nucleotide polymer and the 3'-OH groupfor linking to the next nucleotide. The AZT molecule only contains thefirst of these. Once associated with the HIV enzyme active site, the AZTazide group terminates viral DNA formation because the azide cannot makethe 3',5'-phosphodiester bond with the ribose moiety of the followingnucleoside.

AZT's clinical benefits include increased longevity, reduced frequencyand severity of opportunistic infections, and increased peripheral CD4lymphocyte count. Immunosorbent assays for viral p24, an antigen used totrack HIV-1 activity, show a significant decrease with use of AZT.However, AZT's benefits must be weighed against the adverse reactions ofbone marrow suppression (neutropenia), nausea, myalgia, insomnia, severeheadaches, anemia, and seizures. Furthermore, these adverse side effectsoccur immediately after treatment begins whereas a minimum of six weeksof therapy is necessary to realize AZT's benefits.

Several other nucleotides inhibit HIV reverse transcription as does AZTtriphosphate. Initial tests on 3'-deoxy-3'-fluorothymidine show that itsantiviral activity is comparable to that of AZT. DDC and D4T have beentested in vitro against AZT in a delayed drug administration study; bothwere found to be potent inhibitors of HIV replication with activitiescomparable (D4T) or superior (DDC) to AZT. Both DDC and D4T are inclinical trials. Although DDC is converted to its 5'-triphosphate lessefficiently than its natural analogue, 2'-deoxycytidine, thephosphorylated derivative is resistent to both deaminases andphosphorylases. If dosage and side-effect issues can be resolved, thesedrugs show potential for becoming effective anti-AIDS drugs.

Currently, DDI is used alone or in conjunction with AZT to treat AIDS.However, DDI's side effects include sporadic pancreatitis and peripheralneuropathy. Owing to its toxicity, reduced doses are necessary and thismay limit its usefulness as an antiviral therapeutic treatment. Inaddition, the drug is susceptible to cleavage under acidic conditions.

Recent cell culture tests on BCH-189 have shown that it possessesanti-HIV activity similar to AZT and DDC, but without as much cellulartoxicity. However, BCH-189, like DDC, is toxic at a concentration of ≦10μM in intact CEM cells as measured by cell growth and by determining theextent of mitochondrial DNA synthesis, thus suggesting that one of theside effects of BCH-189 might be clinical peripheral neuropathy.Furthermore, although BCH-189 is less toxic to bone-marrow cells thanAZT, another side effect of BCH-189, like AZT, might be anemia. Thus,there is a need for superior therapeutic agents such as FTC and FTCprodrug analogues that are provided herein. These agents combine highantiviral activity with minimum toxicity for use as inhibitors ofreplication and infectivity of HIV in vivo.

The commonly-used chemical approaches for synthesizing nucleosides ornucleoside analogues can be classified into two broad categories: (1)those which modify intact nucleosides by altering the carbohydrate, thebase, or both and (2) those which modify carbohydrates and incorporatethe base, or its synthetic precursor, at a suitable stage in thesynthesis. Because FTC substitutes a sulfur for a carbon atom in thecarbohydrate ring, only the second approach is applicable. The mostimportant factor in this latter strategy involves delivering the basefrom the β-face of the carbohydrate ring in the glycosylation reactionbecause only the β-isomers exhibit useful biological activity.

It is well known in the art that the stereoselective introduction ofbases to the anomeric centers of carbohydrates can be controlled bycapitalizing on the neighboring group participation of a 2-substituenton the carbohydrate ring [Chem. Ber. 114:1234 (1981)]. However, FTC andits analogues do not possess an exocyclic 2-substituent and, therefore,cannot utilize this procedure unless additional steps to introduce afunctional group that is both directing and disposable are incorporatedinto the synthesis. These added steps would lower the overall efficiencyof the synthesis.

It is also well known in the art that "considerable amounts of theundesired α-nucleosides are always formed during the synthesis of2'-deoxyribosides" [Chem. Ber. 114:1234, 1244 (1981)]. Furthermore, thisreference teaches that the use of simple Friedel-Crafts catalysts likeSnCl₄ in nucleoside syntheses produces undesirable emulsions upon theworkup of the reaction mixture, generates complex mixtures of the α andβ-isomers, and leads to stable α-complexes between the SnCl₄ and themore basic silyated heterocycles such as silyated cytosine. Thesecomplexes lead to longer reaction times, lower yields, and production ofthe undesired unnatural N-3-nucleosides. Thus, the prior art teaches theuse of trimethysilyl triflate or trimethylsilyl perchlorate as acatalyst during the coupling of pyrimidine bases with a carbohydratering to achieve the highest yields of the biologically active β-isomers.However, the use of these catalysts to synthesize FTC or FTC analoguesexhibit little preference for the desired β-isomer; these reactionstypically result in mixtures containing nearly equal amounts of bothisomers. Thus, there exists a need for an efficient synthetic route toFTC and FTC prodrug analogues.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a surprisinglyefficient synthetic route to 2'-deoxy-5-fluoro-3'-thiacytidine (FTC) andvarious FTC prodrug analogues from inexpensive precursors with theoption of introducing functionality as needed. This synthetic routeallows the stereoselective preparation of the biologically active βisomer of these compounds. This invention further relates to thediscovery that FTC and FTC prodrug analogues possess surprisinglysuperior HIV inhibition and cell toxicity effects compared to BCH-189and other analogues of BCH-189, including other 5-halo derivatives ofBCH-189, or other 5-fluoro substituted nucleoside analogues such as2'-deoxy-5-fluoro-3'-oxacytidine (FDOC). Thus, this invention providesfor the therapeutic use of these compounds and pharmaceuticalformulations containing these compounds as antiviral agents.

As used herein, the term "FTC prodrug analogue" refers to a 5'-oxyacylor H substituted and/or 4-N alkyl, substituted alkyl, cyloalkyl or acylsubstituted 2'-deoxy-5-fluoro-3'-thiacytidine that metabolizes to thesame active component or components as FTC. The term "BCH-189 analogues"is meant to refer to nucleosides that are formed from pyrimidine basessubstituted at the 5 position that are coupled to substituted1,3-oxathiolanes.

The synthesis of the present invention includes ozonizing either anallyl ether or ester having the formula CH₂ ═CH--CH₂ --OR or a dietheror diester of 2-butene-1,3-diol having the formula ROCH₂ --CH═CH--CH₂OR, in which R is a protecting group, such as an alkyl, silyl, or acylgroup, to form a glycoaldehyde having the formula OHC--CH₂ --OR; addingthioglycolic acid to the glycoaldehyde to form a lactone of the formula2-(R-oxy)-methyl-5-oxo-1,3-oxathiolane; reducing the lactone to variouscompounds containing a leaving group at the 5 position of theoxathiolane ring; coupling these compounds with a silyated pyrimidinebase fluoro-substituted at the 5 position of the base in the presence ofSNCl₄ to form the β-isomer of a 2'-deoxy-5-fluoro-5'-(R-oxy)-3'-thia-nucleoside analogue; and replacing the R protecting group with ahydrogen or acyl to form FTC or a prodrug analogue of FTC.

Accordingly, one of the objectives of this invention is to provide theantiviral nucleoside B-2'-deoxy-5-fluoro-3'-thiacytidine (FTC), prodruganalogues of FTC that are 5'-oxyacyl substituted and pharmaceuticallyacceptable formulations containing these compounds. Furthermore, it isan object of this invention to provide an efficient and direct methodfor preparing the β-isomer of FTC and prodrug analogues of FTC in highyields. In addition, this invention provides for the use of thesecompounds, or pharmaceutically acceptable formulations containing thesecompounds, as effective and nontoxic antiviral agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates one embodiment of a synthesis of FTC and FTC prodruganalogues according to the present invention;

FIG. 2 illustrates one embodiment of the synthesis of BCH-189 accordingto the present invention;

FIG. 3 illustrates one embodiment of the synthesis of 5-methylcytidineand thymidine derivatives of BCH-189 according to the present invention;

FIG. 4 illustrates one embodiment of the synthesis of BCH-189 andBCH-189 analogues according to the present invention;

FIG. 5 illustrates one embodiment of the synthesis of FDOC, DOC and DOTaccording to the present invention;

FIG. 6 illustrates the effect of delayed treatment on the anti-HIV-1activity of AZT, FTC and other nucleoside analogues in PBM cells;

FIG. 7 illustrates the effect of FTC, BCH-189, DDC and AZT on colonyformation of granulocyte-macrophage precursor cells; and

FIG. 8 illustrates the effect of FTC, AZT and BCH-189 on AZT-resistantand AZT-sensitive HIV-1 in human PBM cells.

DETAILED DESCRIPTION OF THE INVENTION A. Synthesis of FTC or FTC ProdrugAnalogues

FTC is a compound of the formula: ##STR1##

FDOC is a compound of the formula: ##STR2##

Because only the β-isomers of these nucleoside generally exhibit usefulbiological activity, the synthesis for β-FTC is provided for by theinstant invention, using a stereoselective base coupling reaction thatis operative through "in situ" complexation of a suitable cyclicprecursor and Lewis acid. The crucial step in the stereoselectivity ofthe FTC synthesis is the coupling of a2-(R-oxy)-methyl-5-carboxy-1,3-oxathiolane with a silylated pyrimidinebase at ambient temperature using the Lewis acid, SnCl₄. Deprotection ofthe silyl group gives the free nucleoside B-FTC, or its analogues. Theinitial NMR stereochemical assignments have been reconfirmed by X-raystructures, both confirming the β selectivity. Correspondingly, thecrucial step in the stereoselectivity of the FDOC synthesis is thecoupling of a 2-(R-oxy)-methyl-4-carboxy-1,3-dioxolane with a silylatedpyrimidine base at ambient temperature using the Lewis acid, TiCl₄.

Other data regarding these coupling reactions also indicate a metaldependent selectivity. Use of TiCl₄ rather than SnCl₄ in the FTCsynthesis, or SnCl₄ rather than TiCl₄ in the FDOC synthesis, results ina loss in stereoselectivity caused by a Lewis acid-heteroatom mismatch.Furthermore, reactions employing trimethysilyl triflate in bothsyntheses result in non-stereoselective reactions as well.

All of the above results can be rationalized through a heteroatom-Lewisacid interaction. Upon exposure of the carboxylate to the Lewis acid andsilylated base, an intermediate oxonium ion is formed. In the presenceof a complexing Lewis acid, an intermediate could be formed in which themetal would complex to the heteroatom in the ring; one of its ligands,such as chloride or acetate, would be associated with a carbon bearing apartial positive charge. The result of this complexation would beblockage of the α-face opposite to the bulky(t-butyldiphenyl)hydroxymethyl substituent and β attack of the silylatedbase. Use of trimethysilyl triflate or a non-interacting Lewis acidwould generate an oxonium ion that has no facial bias.

A process of the present invention for preparing FTC and FTC prodruganalogues is set forth in FIG. 1. An allyl ether or ester 1 is ozonizedto give an aldehyde 2, which reacts with thioglycolic acid to give alactone 3. The lactone 3 is treated with a reducing agent, followed by acarboxylic anhydride, to produce the carboxylate 4. This carboxylate iscoupled with a silylated 5-fluoro substituted pyrimidine base in thepresence of a Lewis acid that can catalyze stereoselective coupling,such as SnCl₄, to yield the β-isomer of the substituted nucleoside 5 inessentially a 100:0 ratio of β:α isomers. The substituted nucleoside 5is deprotected to produce FTC 6 or modified at the 5'- position to forma FTC prodrug analogue.

The process for preparing FDOC is set forth in FIG. 5. Glycolic acidreacts with glycoaldehyde 9 to form the lactone 28, which is reduced toform the carboxylate 29. 29 is coupled with a silylated 5-fluorosubstituted pyrimidine base in the presence of a Lewis acid that cancatalyze stereoselective coupling, such as TiCl₄, TiCl₃ (OiPr) or TiCl₂(OiPr)₂, to yield the β-isomer of the substituted nucleoside 36. Thesubstituted nucleoside 36 is deprotected to produce FDOC 37.

The protecting group R in 1 can be selected to provide protection forthe corresponding alcohol until the final step in the synthesis iscarried out (deprotection of 5 to form 6). Any group that functions inthis manner may be used. For instance, alkyl, silyl, and acyl protectinggroups or groups that possess substantially the same properties as thesegroups can be used.

An alkyl protecting group, as used herein, means triphenylmethyl or analkyl group that possesses substantially the same protecting propertiesas triphenylmethyl. A silyl protecting group, as used herein, means atrialkylsilyl group having the formula: ##STR3## wherein R₁, R₂, and R₃may be lower-alkyl, e.g., methyl, ethyl, butyl, and alkyl possessing 5carbon atoms or less; or phenyl. Furthermore, R may be identical to R₂ ;R₁, R₂, and R₃ may all be identical. Examples of silyl protecting groupsinclude, but are not limited to, trimethylsilyl andt-butyldiphenylsilyl.

An acyl group, as used herein to describe an acyl protecting group (asin 1) or to describe a carboxylate (as in 4), is a group having theformula: ##STR4## wherein R' is a lower alkyl, e.g., methyl, ethyl,butyl, and alkyl possessing 5 carbon atoms or less; substituted loweralkyl wherein the alkyl bears one, two, or more simple substituents,including, but not limited to, alkyl, amino, carboxyl, pavoloyl,hydroxy, phenyl, lower-alkoxy, e.g., methoxy and ethoxy; phenyl;substituted phenyl wherein the phenyl bears one, two, or more simplesubstituents, including, but not limited to, lower alkyl, halo, e.g.,chloro and bromo, sulfato, sulfonyloxy, carboxyl, carbo-lower-alkoxy,e.g., carbomethoxy and carbethoxy, amino, mono- and di-lower alkylamino,e.g., methylamino, amido, hydroxy, lower alkoxy, e.g., methoxy andethoxy, lower-alkanoyloxy, e.g., acetoxy.

A 5-fluoro substituted silyated pyrimidine base, as used herein, means acompound having the formula: ##STR5## wherein X is either atrialkylsilyloxy or a trialkylsilylamino group and Z is a trialkylsilylgroup. A trialkylsilyl group, as used herein, means a group having theformula: ##STR6## wherein R₁, R₂, and R₃ may be lower-alkyl, e.g.,methyl, ethyl, butyl, and alkyl possessing 5 carbon atoms or less, orphenyl. Furthermore, R₁ may be identical to R₂ ; R₁, R₂, and R₃ may allbe identical. Examples of trialkylsilyl groups include, but are notlimited to, trimethylsilyl and t-butyldiphenylsilyl.

As used herein, a leaving group means a functional group that forms anincipient carbocation when it leaves.

Illustrative examples of the synthesis of FTC or FTC prodrug analogues,BCH-189 or BCH-189 analogues and FDOC according to the present inventionare given in FIGS. 1-5 and Examples 1-6.

EXAMPLE 1--SYNTHESIS OF BCH-189

FIG. 2 shows the synthesis of BCH-189 starting with allyl alcohol 7. ANaH oil suspension (4.5 g, 60%, 110 mmol) was washed with THF twice (100ml×2) and the resulting solid suspended in THF (300 ml). The suspensionwas cooled to 0° C., allyl alcohol 7 (6.8 ml, 100 mmol) was addeddropwise, and the mixture was stirred for 30 minutes at 0° C.t-Butyl-diphenylsilyl chloride (25.8 ml, 100.8 mmol) was added dropwiseat 0° C. and the reaction mixture was stirred for 1 hour at 0° C. Thesolution was quenched with water (100 ml), and extracted with diethylether (200 ml×2). The combined extracts were washed with water, driedover MgSO₄, filtered, concentrated, and the residue distilled undervacuum (90°-100° C. at 0.5-0.6 mm Hg) to give a colorless liquid 8 (28g., 94 mmol, 94%). (¹ H NMR: (CDCl₃, 300 MHz) 7.70-7.35 (10H, m,aromatic-H); 5.93 (1H, m, Hz); 5.37 (1H, dt, H₁) J=1.4 and 14.4 Hz; 5.07(1H, dt, H₁) J=1.4 and 8.7 Hz; 4.21 (2H, m, H₃); 1.07 (9H, s, t-Bu))

The silyl allyl ether 8 (15.5 g, 52.3 mmol) was dissolved in CH₂ Cl₂(400 ml), and ozonized at -78° C. Upon completion of ozonolysis, DMS (15ml, 204 mmol, 3.9 eq) was added at -78° C. and the mixture was warmed toroom temperature and stirred overnight. The solution was washed withwater (100 ml×2), dried over MgSO₄, filtered, concentrated, anddistilled under vacuum (100°-110° C. at 0.5-0.6 mm Hg) to give acolorless liquid 9 (15.0 g, 50.3 mmol, 96%). (¹ H NMR: (CDCl₃, 300 MHz)9.74 (1H, s, H-CO); 7.70-7.35 (10H, m, aromatic-H); 4.21 (2H, s, -CH₂);1.22 (9H, s, t-Bu))

Silylated glycoaldehyde 9 (15.0 g, 50.3 mmol) was dissolved in toluene(200 ml) and thioglycolic acid (3.50 ml, 50.3 mmol) was added all atonce. The solution was refluxed for 2 hours while the resulting waterwas removed with a Dean-Stark trap. The solution was cooled to roomtemperature and washed with saturated NaHCO₃ solution and the aqueouswashings were extracted with diethyl ether (200 ml×2). The combinedextracts were washed with water (100 ml×2), dried over MgSO₄, filtered,and concentrated to give a colorless oil 10 (16.5 g, 44.3 mmol, 88%),which gradually solidified under vacuum. Recrystallization from hexaneafforded a white solid 10 (15.8 g, 84%). (¹ H NMR: 7.72-7.38 (10H, m,aromatic-H); 5.53 (1H, t, H₂) J=2.7 Hz; 3.93 (1H, dd, --CH₂ O) J=9.3 Hz;3.81 (1H, d, 1H₄ ) J=13.8 Hz; 3.79 (1H, dd, --CH₂ O); 3.58 (1H, d, 1H₄);1.02 (9H, s, t-Bu))

2-(t-Butyl-diphenylsilyloxy)-methyl-5-oxo-1,2-oxathiolane 10 (5.0 g,13.42 mmol) was dissolved in toluene (150 ml) and the solution wascooled to -78° C. Dibal-H solution (14 ml, 1.0 M in hexanes, 14 mmol)was added dropwise, while the inside temperature was kept below -70° C.all the time. After the completion of the addition, the mixture wasstirred for 30 minutes at -78° C. Acetic anhydride (5 ml, 53 mmol) wasadded and the mixture was warmed to room temperature and stirredovernight. Water (5 ml) was added to the mixture and the resultingmixture was stirred for 1 hour at room temperature. The mixture wasdiluted with diethyl ether (300 ml), MgSO₄ (40 g) was added, and themixture was stirred vigorously for 1 hour at room temperature. Themixture was filtered, concentrated, and the residue flashchromatographed with 20% EtOAc in hexanes to give a colorless liquid 11(3.60 g, 8.64 mmol, 64%), which was a 6:1 mixture of anomers. (¹ H NMRof the major isomer: 7.70-7.35 (10H, m, aromatic-H); 6.63 (1H, d, H₅)J=4.4 Hz; 5.47 (1H, t, H₂); 4.20-3.60 (2H, m, --CH₂ O); 3.27 (1H, dd,1H₄) J=4.4 and 11.4 Hz; 3.09 (1H, d, 1H₄) J=11.4 Hz; 2.02 (3H, s, CH₃CO); 1.05 (9H, s, tBu); ¹ H NMR of the minor isomer: 7.70-7.35 (10H, m,aromatic-H); 6.55 (1H, d, H₅) J=3.9 Hz; 5.45 (1H, t, Hz); 4.20-3.60 (2H,m, --CH₂ O); 3.25 (1H, dd, 1H₄) J=3.9 and 11.4 Hz; 3.11 (1H, d, 1H₄)J=11.4 Hz; 2.04 (3H, s, CH₃ CO); 1.04 (9H, s, t-Bu))

Alternatively, 50 g (0.134 mol, 1.0 eq) of2-(t-Butyldiphenylsilyloxy)-methyl-5-oxo-1,2-oxathiolane 10 in 500 ml ofanhydrous tetrahydrofuran was transferred into a flame-dried,argon-charged 3,000 ml three-necked round-bottomed flask, equipped withan addition funnel and thermometer. The clear solution was cooled to-10° C. (ice/acetone bath) and treated with 147 ml (0.147 mol, 1.1equiv) of a 1 M solution of lithium tri-t-butoxy aluminum hydride in THF(prepared solution of the solid obtained from Aldrich). The reaction wasqualitatively monitored for the disappearance of the lactone (R_(f)=0.38) and the appearance of a second UV-active component at R_(f) =0.09(SiO₂, eluting with 90% hexanes in ethyl acetate). In addition, thereaction was quantitatively monitored by GC. The lactol formed wasallowed to react at room temperature with 126 ml (1.34 mol, 10.0 equiv)of acetic anhydride (freshly distilled from calcium hydride). Thereaction was monitored by the appearance of UV-active component at R_(f)=0.34 (SiO₂, eluting with 90% hexanes in ethyl acetate) and GC until nolactol was detected. The reaction was quenched with saturated sodiumbicarbonate solution and stirred overnight. Anhydrous magnesium sulfatewas added and the resulting mixture filtered, concentrated and placedunder vacuum to give 49.3 g of crude material 11 as a light red oil.

2-(t-Butyl-diphenylsilyloxy)-methyl-5-acetoxy-1,3-oxathiolane 11 (0.28g, 0.67 mmol) was dissolved in 1,2-dichloroethane (20 ml), and silylatedcytosine 12 (0.20 g, 0.78 mmol) was added at once at room temperature.The mixture was stirred for 10 minutes and to it was added SnCl₄solution (0.80 ml, 1.0 M solution in CH₂ Cl₂, 0.80 mmol) dropwise atroom temperature. Additional cytosine 12 (0.10 g, 0.39 mmol) and SnCl₄solution (0.60 ml) were added in a same manner 1 hour later. Aftercompletion of the reaction in 2 hours, the solution was concentrated,and the residue was triturated with triethylamine (2 ml) and subjectedto flash chromatography (first with neat EtOAc and then 20% ethanol inEtOAc) to give a tan solid 13 (100% B configuration) (0.25 g, 0.54 mmol,80%). (¹ H NMR (DMSO-d⁶): 7.75 (1H, d, H₆) J= 7.5 Hz; 7.65-7.35 (10H, m,aromatic-H); 7.21 and 7.14 (2H, broad, --NH₂); 6.19 (1H, t, H_(5'));5.57 (1H, d, H₅); 5.25 (1H, t, H_(2')); 3.97 (1H, dd, --CH₂ O) J=3.9 and11.1 Hz: 3.87 (1H, dd, --CH₂ O): 3.41 (1H, dd, 1H_(4')) J=4.5 and 11.7HZ; 3.03 (1H, dd, 1H_(4')) J=?; 0.97 (9H, s, t-Bu))

Silyether 13 (0.23 g, 0.49 mmol) was dissolved in THF (30 ml), and to itwas added n-Bu₄ NF solution (0.50 ml, 1.0 M solution in THF, 0.50 mmol)dropwise at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up withethanol/triethylamine (2 ml/1 ml), and subjected to flash chromatography(first with EtOAc, then 20% ethanol in EtOAc) to afford a white solid 14in 100% anomeric purity (BCH-189; 0.11 g, 0.48 mmol, 98%), which wasfurther recrystallized from ethanol/CHCl₃ /Hexanes mixture. (¹ H NMR(DMSO-d₆): 7.91 (1H, d, H₆) J=7.6 Hz; 7.76 and 7.45 (2H, broad--NH₂);6.19 (1H, t, H_(5')); 5.80 (1H, d, H₅) J=7.6 Hz; 5.34 (1H, broad, --OH);5.17 (1H, t, H_(2')); 3.74 (2H, m, --CH₂ O); 3.42 (1H, dd, 1H_(4'))J=5.6 and 11.5 Hz; 3.09 (1H, dd, 1H_(4')) J=4.5 and 11.5 Hz)

EXAMPLE 2--SYNTHESIS OF BCH-189 FROM A URACIL DERIVATIVE

BCH-189 and its analogues can also be synthesized by coupling asilylated uracil derivative with 11. Silylated uracil derivative 15(1.80 g, 7.02 mmol) was coupled with 11 (1.72 g, 4.13 mmol) in1,2-dichloroethane (50 ml) in the presence of SnCl₄ (5.0 ml) asdescribed above in the preparation of the cytosine derivative 13. Thereaction was complete after 5 hours. Flash chromatography, first with40% EtOAc in hexane and then EtOAc, afforded a white foam 16 (1.60 g,3.43 mmol, 83%). (¹ H NMR: 9.39 (1H, broad, --NH) 7.90 (1H, d, H₆) J=7.9Hz; 7.75-7.35 (10H, m, aromatic-H); 6.33 (1H, dd, H_(5')); 5.51 (1H, d,H₅) J=7.9 Hz; 5.23 (1H, t, H_(2')); 4.11 (1H, dd, --CH₂ O) J=3.2 and11.7 Hz; 3.93 (1H, dd, --CH₂ O); 3.48 (1H, dd, 1H_(4')) J=5.4 and 12.2Hz; 3.13 (1H, dd, 1H_(4')) J=3.2 and 12.2 Hz)

The uracil derivative 16 can be converted to the cytosine derivative 13.The uracil derivative 16 (0.20 g, 0.43 mmol) was dissolved in a mixtureof pyridine/dichloroethane (2 ml/10 ml), and the solution cooled to 0°C. Triflic anhydride (72 μl, 0.43 mmol) was added dropwise at 0° C. andthe mixture was warmed to room temperature and stirred for 1 hour.Additional triflic anhydride (0.50 μl, 0.30 mmol) was added and themixture stirred for 1 hour. TLC showed no mobility with EtOAc. Thereaction mixture was then decannulated into a NH₃ -saturated methanolsolution (30 ml) and the mixture wa stirred for 12 hours at roomtemperature. The solution was concentrated, and the residue subjected toflash chromatography to give a tanned foam 13 (0.18 g, 0.39 mmol, 91%),which was identical with the compound obtained from the cytosinecoupling reaction.

EXAMPLE 3--SYNTHESIS OF 5-METHYLCYTIDINE AND THYMIDINE BCH-189DERIVATIVES

FIG. 3 illustrates the synthesis of 5-methylcytidine and thymidinederivatives of BCH-189. The acetate 11 (0.93 g, 2.23 mmol) in1,2-dichloroethane (50 ml), was reacted with the silylated thyminederivative 17 (1.0 g, 3.70 mmol), and SnCl₄ solution (4.0 ml) in amanner similar to that described for the preparation of cytosinederivative 13. (¹ H NMR: 8.10 (1H, broad, NH); 7.75-7.30 (11H, m, 10Aromatic H's and 1H₆); 6.32 (1H, t, H_(1')) J=5.4 Hz; 5.25 (1H, t,H_(4')) J=4.2 Hz; 4.01 (1H, dd, 1H_(5')) J=3.9 and 11.4 Hz; 3.93 (1H,dd, 1H_(5')) J=4.5 and 11.4 Hz; 3.41 (1H, dd, 1H_(2')) J=5.4 and 11.7Hz; 3.04 (1H, dd, 1H_(2')) J=5.7 and 11.7 Hz; 1.75 (3H, s, CH₃); 1.07(9H, s, t-Bu))

The thymine derivative 18 (0.20 g, 0.42 mmol) was dissolved in a mixtureof pyridine/dichloroethane (2 ml/10 ml), and the solution cooled to 0°C. To it was added triflic anhydride (100 μl, 0.60 mmol) dropwise at 0°C., and the mixture was allowed, with continuous stirring, to warm toroom temperature. After reaching room temperature, it was stirred for 1hour. TLC showed no mobility with EtOAc. The reaction mixture was thendecannulated into the NH₃ -saturated methanol solution (20 ml), and themixture stirred for 12 hours at room temperature. The solution wasconcentrated, and the residue was subjected to flash chromatography togive a tanned foam 19 (0.18 g, 0.38 mmol, 90%). (¹ H NMR: 7.70-7.30(12H, m, 10 Aromatic H's, 1NH and H₆); 6.60 (1H, broad, 1NH); 6.34 (1H,t, H_(1')) J=4.5 Hz; 5.25 (1H, t, H_(4')) J=3.6 H₂ ; 4.08 (1H, dd,1H_(5')) J=3.6 and 11.4 Hz; 3.96 (1H, dd, 1H_(5')) J=3.6 and 11.4 Hz;3.52 (1H, dd, 1H_(2')) J=5.4 and 12.3 Hz; 3.09 (1H, dd, 1H_(2')) J=3.9and 12.3 Hz; 1.72 (3H, s, CH₃); 1.07 (9H, s, t-Bu))

Silylether 19 (0.18 g, 0.38 mmol) was dissolved in THF (20 ml), and ann-Bu₄ NF solution (0.50 ml, 1.0 M solution in THF, 0.50 mmol) was added,dropwise, at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up withethanol/triethylamine (2 ml/1 ml), and subjected to flash chromatography(first with EtOAc, then 20% ethanol in EtOAc) to afford a white solid 20(0.09 g, 0.37 mmol, 97%), which was further recrystallized fromethanol/CHCl₃ /Hexanes mixture to afford 82 mg of pure compound (89%).(¹ H NMR: (in d⁶ -DMSO): 7.70 (1H, s, H₆); 7.48 and 7.10 (2H, broad,NH₂); 6.19 (1H, t, H_(1')) J=6.5 Hz; 5.31 (1H, t, OH); 5.16 (1H, t,1H_(4')) J=5.4 Hz; 3.72 (2H, m, 2H_(5')) 3.36 (1H, dd, 1H_(2')) J=6.5and 14.0 Hz; 3.05 (1H, dd, 1H_(2')) J=6.5 and 14.0 Hz; 1.85 (3H, s,CH₃))

Silylether 18 (0.70 g, 1.46 mmol) was dissolved in THF (50 ml), and ann-Bu₄ NF solution (2 ml, 1.0 M solution in THF, 2 mmol) was added,dropwise, at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up withethanol/triethylamine (2 ml/1 ml), and subjected to flash chromatographyto afford a white solid 21 (0.33 g, 1.35 mmol, 92%). (¹ H NMR: (in d⁶-Acetone): 9.98 (1H, broad, NH); 7.76 (1H, d, H₆) J=1.2 Hz; 6.25 (1H, t,H_(4')) J=5.7 Hz; 5.24 (1H, t, H_(1')) J=4.2 Hz; 4.39 (1H, t, OH) J=5.7Hz; 3.85 (1H, dd, 2H_(5')) J=4.2 and 5.7 Hz; 3.41 (1H, dd, 1H_(2'))J=5.7 and 12.0 Hz; 3.19 (1H, dd, 1H_(2')) J=5.4 and 12.0 Hz; 1.80 (3H,s, CH₃))

EXAMPLE 4--SYNTHESIS OF FTC

Acetate 11 (1.70 g, 4.08 mmol) was dissolved in dichloromethane (100ml). Silylated 5-fluorocytosine (1.22 g, 4.5 mmol) was mixed with tin(IV) chloride solution (8.6 ml, 1.0 M in dichloromethane, 8.6 mmol) indichloromethane (20 ml). The pre-mixed solution was decannulated in theacetate solution over 20 minutes. The mixture was stirred for 3 hours atroom temperature, then pyridine (3 ml) was added to the mixture in oneportion. The mixture was concentrated under vacuum, and the residuetaken up with ethanol (10 ml) and subjected to flash chromatography togive a tan solid (1.80 g, 3.71 mmol, 91%), which was furtherrecrystallized from ethanol to give a total of 1.75 g of a crystallinecompound(5'-O-t-Butyldiphenysilyl-3'-thia-2',3'-dideoxy-5-fluorocytidine, 100% Bconfiguration). (¹ H NMR: (DMSO-d⁶) 7.96 (1H, d, H₆, J=6.8 Hz), 7.87 &7.61 (2H, broad, NH₂), 7.64 & 7.43 (10H, m, Aromatic H's), 6.19 (1H, t,H_(1'), J=5.4 Hz), 5.28 (1H, t, H_(4'), J=4.0 Hz), 4.01 (H, dd, 1H_(5'), J=3.6 & 11.5 Hz), 3.90 (1H, dd, 1H_(5'), J=4.3 & 11.5 Hz), 3.45(1H, dd, 1H_(2'), J=5.4 & 11.5 Hz), 3.16 (1H, dd, 1H_(2'), J=5.4 & 11.5Hz); mp 214°-215° C.; Anal. Calc. for C₂₄ H₂₈ O₃ N₃ FSSi: C, 59.36; H,5.81; N, 8.65; S, 6.60. Found: C, 59.44; H, 5.81; N, 8.60; S, 6.64.

The silylether(5'-O-t-Butyldiphenysilyl-3'-thia-2',3'-dideoxy-5-fluorocytidine, 100% βconfiguration)(1.12 g, 2.31 mmol) was dissolved in THF (80 ml), and toit was added n-Bu₄ NF solution (2.50 ml, 1.0 M solution in THF, 2.50mmol) dropwise at room temperature. The mixture was stirred for 0.5hours and concentrated under vacuum. The residue was taken up withEtOH/pyridine (3 ml/1 ml), and subjected to flash chromatography toafford a white solid (0.75 g), which was further recrystallized fromEtOH to give a total of 0.56 g of the crystalline compound2'-Deoxy-5-fluoro-3'-thiacytidine (FTC; 100% β isomer; 2.26 mmol; 98%).(1H NMR: (DMSO-d⁶) 8.18 (1H, d, H₆, J=8.4 Hz), 7.81 & 7.57 (2H, broad,NH₂), 6.12 (1H, dd, H_(1'), J=5.7 & 4.2 Hz), 5.40 (1H, t, OH, J=5.7 Hz),5.17 (1H, t, H_(4'), J=3.6 Hz), 3.74 (2H, m, 2H_(5')), 3.41 (1H, dd,1H_(2'), J=5.7 & 11.7 Hz), 3.11 (1H, dd, 1H_(2'), J=4.2 & 11.7 Hz); ¹³ CNMR: (DMSO-d⁶) 157.85 (d, J=13.4 Hz), 153.28, 136.12 (d, J=241 Hz),126.01 (d, J=32.6 Hz), 86.90, 86.84, 62.48, 37.07; mp 195°-196° C.;Anal. Calc. for C₈ H₁₀ O₃ N₃ SF: C, 38.86; H, 4.08; N, 17.00; S, 12.97.Found: C, 38.97; H, 4.07; N, 16.93; S, 12.89.)

EXPERIMENT 5--SYNTHESIS OF 5-HALO DERIVATIVES OF β-BCH-189

The coupling of the acetate 11 with various bases was done as shown inFIG. 4. This coupling could be done, in general, in two ways to obtainthe cytidine analogues, either by direct coupling of the acetate with acorresponding bis-silylated cytosines in the presence of tin(IV)chloride or by ammonolysis of the triflate derived from thecorresponding uridine analogues. The typical experimental procedure isoutlined below.

The acetate 25 (0.28 g, 0.67 mmol) was dissolved in 1,2-dichloroethane(20 ml), and to it the silylated cytosine (0.20 g, 0.78 mmol) was addedin one portion at room temperature. The mixture was stirred for 10minutes and to it a SnCl₄ solution (1.34 ml, 1.0 M solution in CH₂ Cl₂,1.34 mmol) was added, dropwise, at room temperature. Upon completion,the solution was concentrated, the residue was triturated with Et₃ N (2ml) and subjected to flash chromatography to give a tan solid 26 (0.25g, 0.54 mmol, 80%).

Silylether 26 (0.23 g, 0.49 mmol) was dissolved in THF (30 ml), and ann-Bu₄ NF solution (0.50 ml, 1.0 M solution in THF, 0.50 mmol) was added,dropwise, at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up with EtOH/Et₃ N (2ml/1 ml), and subjected to flash chromatography to afford a white solid27 (100% B isomer; 0.11 g, 0.48 mmol, 98%), which Was furtherrecrystallized from EtOH/CHCl₃ /Hexanes mixture.

The procedure for coupling a silylated uracil with acetate 25 is asfollows: The acetate 25 (1.72 g, 4.13 mmol), in 1,2-dichloroethane (50ml), was reacted with the silylated uracil derivative (1.80 g, 7.02mmol) and SnCl₄ solution (5.0 ml) for 5 hours to complete the reaction.Flash chromatography with 40% EtOAc in hexane and then EtOAc afforded awhite foam 26 (1.60 g, 3.43 mmol, 83%).

The uracil derivative 26 (0.20 g, 0.43 mmol) was dissolved in a mixtureof pyridine/dichloroethane (2 ml/10 ml), and the solution cooled to 0°C. To the solution was added Tf₂ O (72 μl, 0.43 mmol) dropwise at 0° C.and the mixture was allowed, with continuous stirring, to warm to roomtemperature. After reaching room temperature, it was stirred for 1 hour.Additional Tf₂ O (0.50 μl, 0.30 mmol) was added and the mixture wasstirred for 1 hour. TLC showed no mobility with EtOAc. The reactionmixture was then decannulated into the NH₃ -saturated methanol solution(30 ml), and the mixture stirred for 12 hours at room temperature. Thesolution was concentrated and the residue was subjected to flashchromatography to give a tanned foam 27 (100% β isomer; 0.18 g, 0.39mmol, 91%), which was identical with the compound obtained from thecytosine coupling reaction.

The compounds synthesized include:

2'Deoxy5-methyl-3'-thiacytidine: ¹ H NMR (DMSO-d⁶) 7.70 (1H, s, H₆),7.48 and 7.10 (2H, broad, NH₂), 6.19 (1H, t, H_(1'), J=5.4 Hz), 5.31(1H, t, OH, J=4.5 Hz), 5.16 (1H, t, H_(4'), J=4.5 Hz), 3.72 (2H, m,2H_(5')), 3.36 (1H, dd, 1H_(2'), J=5.4 & 11.7 Hz), 3.05 (1H, dd,1H_(2'), J=5.4 & 11.7 Hz), 1.85 (3H, d, CH₃, J_(allylic) =0.6 Hz); mp183°-185° C.

2'-Deoxy-5-fluoro-3'-thiacytidine: ¹ H NMR (DMSO-d⁶) 8.18 (1H, d, H₆,J=8.4 Hz), 7.81 & 7.57 (2H, broad, NH₂), 6.12 (1H, dd, H_(1'), J=5.7 &4.2 Hz), 5.40 (1H, t, OH, J=5.7 Hz), 5.17 (1H, t, H_(4'), J=3.6 Hz),3.74 (2H, m, 2H_(5')), 3.41 (1H, dd, 1H_(2'), J=5.7 & 11.7 Hz), 3.11(1H, dd, 1H_(2'), J=4.2 & 11.7 Hz); mp 195°-196° C.; Anal. Calc. for C₈H₁₀ O₃ N₃ SF: C, 38.86; H, 4.08; N, 17.00; S, 12.97. Found: C, 38.97; H,4.07; N, 16.93; S, 12.89.

2'-Deoxy-5-chloro-3'-thiacytidine: ¹ H NMR (DMSO-d⁶) 8.30 (1H, s, H₆),7.89 & 7.26 (2H, broad, NH₂), 6.13 (1H, t, H_(1'), J=4.5 Hz), 5.45 (1H,t, OH, J=5.7 Hz), 5.19 (1H, t, H_(4'), J=3.6 Hz), 3.76 (2H, m, 2H_(5')),3.44 (1H, dd, 1H_(2'), J=5.4 & 12.0 Hz), 3.16 (1H, dd, 1H_(2'), J=3.9 &12.0 Hz); mp 212°-212.5° C.; Anal. Calc. for C₈ H₁₀ O₃ N₃ SCl: C, 36.44;H, 3.82; N, 15.93; S, 12.16; Cl, 13.44. Found: C, 36.53; H, 3.86; N,15.90; S, 12.08; Cl, 13.50.

2'-Deoxy-5-bromo-3'-thiacytidine: ¹ H NMR (DMSO-d⁶) 8.37 (1H, s, H₆),7.90 & 7.05 (2H, broad, NH₂), 6.14 (1H, t, H_(1'), J= 4.5 Hz), 5.46 (1H,t, OH, J=5.4 Hz), 5.19 (1H, t, H_(4'), J=3.6 Hz), 3.76 (2H, m, 2H_(2')),3.41 (1H, dd, 1H_(2'), J=5.4 & 12.0 Hz), 3.16 (1H, dd, 1H_(2'), J=3.6 &12.0 Hz); mp 197°-198° C.; Anal. Calc. for C₈ H₁₀ O₃ N₃ SBr: C, 331.18;H, 3.27; N, 13.64; S, 10.40; Br, 25.93. Found: C, 31.29; H, 3.29; N,13.54; S, 10.49; Br, 25.98.

2'-Deoxy-5-iodo-3'-thiacytidine: ¹ H NMR (DMSO-d⁶) 8.36 (1H, s, H₆),7.87 & 6.66 (2H, broad, NH₂), 6.13 (1H, t, H_(1'), J=4.5 Hz), 5.44 (1H,t, OH, J=5.7 Hz), 5.18 (1H, t, H_(4'), J=3.6 Hz), 3.73 (2H, m, 2H_(5')),3.42 (1H, dd, 1H_(2'), J=5.7 & 12.0 Hz), 3.14 (1H, dd, 1H_(2'), J=3.6 &12.0 Hz); mp 188°-189° C.

2'-Deoxy-5-fluoro-3'-thiauridine: ¹ H NMR (DMSO-d⁶) 11.89 (1H, broad,NH), 8.33 (1H, d, H₆, J=7.5 Hz), 6.15 (1H, t, H_(1'), J=3.9 Hz), 5.44(1H, t, OH, J=5.7 Hz), 5.19 (1H, t, H_(4'), J=3.6 Hz), 3.75 (2H, m,2H_(5')), 3.43 (1H, dd, 1H_(2'), J=5.7 & 12.0 Hz), 3.25 (1H, dd,1H_(2'), J=4.2 & 12.0 Hz); mp 158°-159° C.; Anal. Calc. for C₈ H₉ O₄ N₂SF: C, 38.71; H, 3.65; N, 11.29; S, 12.92. Found: C, 38.79; H, 3.68; N,11.23; S, 12.82.

2'l -Deoxy-5-chloro-3'-thiauridine: ¹ H NRM (DMSO-d⁶) 11.95 (1H, broad,NH), 8.11 (1H, s, H₆), 6.18 (1H, t, H_(1'), J=4.8 Hz), 5.38 (1H, t, OH,J=3.6 Hz), 4.47 (1H, dd, 1H_(5'), J=4.5 % 12.3 Hz), 4.37 (1H, dd,1H_(5'), J=3.0 & 12.3 Hz), 3.49 (1H, dd, 1H_(2'), J=5.4 & 12.0 Hz), 3.38(1H, dd, 1H_(2'), J=4.2 & 12.0 Hz).

2'-Deoxy-5-iodo-3'-thiauridine: ¹ H NMR (DMSO-d⁶) 11.73 (1H, broad, NH),8.48 (1H, s, H₆), 6.15 (1H, dd, H_(1'), J=4.0 & 5.0 Hz), 5.46 (1H, t,OH, J=5.4 Hz), 5.19 (1H, t, H_(4'), J=3.6 Hz), 3.76 (2H, m, 2H_(5')),3.44 (1H, dd, 1H_(2'), J=5.4 & 12.0 Hz), 3.30 (1H, dd, 1H_(2'), J=4.7 &12.0 Hz); mp 177°-179° C.

EXAMPLE 6--SYNTHESIS OF DOC, DOT, and FDOC

FIG. 5 shows the synthesis of 2'-deoxy-3'-oxacytidine (DOC),2'-deoxy-3'-oxathymidine (DOT), and 2'-deoxy-5-fluoro-3'-oxacytidine(FDOC) according to the present invention. The silyated glycoaldehyde 9was prepared as in Example 1. (4.0 g, 13.40 mmol) of 9 was dissolved in1,2-dichloroethane (50 ml) and to it was added glycolic acid (1.10 g,14.46 mmol) in one portion and p-toluenesulfonic acid (0.1 g). Themixture was refluxed for 1 hour. The volume of the solution was thenreduced to about half by distilling off the solvent with a Dean-Starktrap. Another 50 ml of dichloroethane was added and the solutionrefluxed for 30 minutes again. The solution was cooled to roomtemperature and concentrated under vacuum. The residue was dissolved inether (200 ml) and the solution washed with NaHCO₃ solution (50 ml) andwater (50 ml). The combined extracts were dried over MgSO₄, filtered,and concentrated to give a colorless oil which gradually solidifiedunder vacuum. Recrystallization from hexane afforded a waxy white solid28 (2-(t-Butyldiphenylsilyloxy)-methyl-4-oxo-1,3-dioxolane) (4.2 g,11.78 mmol, 88%). (¹ H NMR : (CDCl₃, 300 MHz) 7.66 & 7.42 (10H, m,aromatic-H), 5.72 (1H, broad, Hz), 4.46 (1H, d, 1H₅, J=14.4 Hz), 4.28(1H, d, 1H₅, J=14.4 Hz), 3.81 (2H, d, 2CH₂ O, J=1.8 Hz), 1.04 (9H, s,t-Bu); mp 94°-95° C.; MS (FAB) 357 (M+H), 299, 241, 197, 163, 135, 91;Anal. Calc'd for C₂₀ H₂₄ O₄ Si: C, 67.38; H, 6.79; Found: C, 67.32; H,6.77.)

4-Acetoxy-2-(t-Butyldiphenylsilyloxymethyl)-1,3-dioxolane 29 wasprepared using either of the following procedures A or B.

Procedure A: (DIBAL-H) The lactone 28 (1.0 g, 2.81 mmol) was dissolvedin toluene (100 ml), and the solution cooled to -78° C. Dibal-H solution(3.0 ml, 1.0 M in hexanes, 3 mmol) was added dropwise, while the insidetemperature was kept below -70° C. throughout the addition. After theaddition was completed, the mixture was stirred for 0.5 hours at -78° C.To it was added Ac₂ O (5 ml, 53 mmol) and the mixture, with continuousstirring, was allowed to reach room temperature overnight. Water (5 ml)was added to it and the mixture was stirred for 1 h, MgSO₄ (40 g) wasthen added, and the mixture was stirred vigorously for 1 hour at roomtemperature. The mixture was filtered, concentrated, and the residueflash chromatographed with 20% EtOAc in hexanes to give a colorlessliquid 29 (0.70 g) which was a mixture of the desired acetates and thealdehyde 9 derived from the ring opening reaction.

Procedure B: (LiAlH(OtBu)₃) Lactone 28 (1.426 g, 4 mmol) was dissolvedin 20 ml of THF, cooled to 0° C., and to this was added 5 ml (5 mmol,1.25 eq) of a LiAlH(OtBu)₃ solution (1 M in THF; Aldrich) over a 40minute period. After addition was completed, the mixture was stirred for6 hours at 0° C. After this time, 3.8 ml (40 mmol, 10 eq) of dry aceticanhydride was added, and the mixture was warmed to room temperature. Thereaction was then stirred for another 40 hours and then was quenched byadding 50 ml of ether and 50 ml of saturated NaHCO₃ solution. The layerswere separated after 2 hours of stirring, and the organic layer waswashed successively with saturated NaHCO₃ and NaCl solutions. Theaqueous layers were combined and then re-extracted with 75 ml of ether(3 times). The organic layers were combined, dried over MgSO₄, filtered,and the solvent was removed. Column chromatography (Hexanes/EtOAc, 6/1)gave 1.09 g, which was 69% (753 mg, 47% yield) of the desired acetates29 (3.6:1 ratio at the glycosidic center) by ¹ H NMR analysis (the restof the mixture was composed of the aldehyde 9 and the lactone 28, whichwere difficult to separate).

(¹ H NMR: (CDCl₃, 300 MHz) 1.02 (s, 9H, major isomer), 1.04 (s, 9H,minor isomer), 1.96 (s, 3H, minor), 2.12 (s, 3H, major), 3.7 (m, 2H),4.07 (m, 2H), 5.24 (t, 1H, minor, J=4.2 Hz), 5.37 (t, 1H, major, J=3Hz), 6.3 (t, 1H, minor, J=3.9 Hz), 6.37 (dd, 1H, major, J=1.5 Hz, J=4.5Hz), 7.39 (m, 6H), 7.67 (m, 4H). IR (neat): cm⁻¹ 3090, 2980, 2880, 1760,1475, 1435, 1375, 1240, 1120, 1000. MS (FAB, Li⁺): 407(M+Li), 312, 282,241, 197, 162, 125. Anal. Calc. for C₂₂ H₂₈ O₅ Si: C, 65.97%, H, 7.05%;Found: C, 66.60%, H, 7.27%.)

The crude acetate 29 (0.25 g, 0.62 mmol, quantity assumed with 0.50 g ofthe previous mixture) was dissolved in methylene chloride (50 ml), andto it the silylated cytosine 30 (X=H) (0.10 g, 0.63 mmol) was added inone portion. The mixture was stirred for 10 minutes, and to it a TiCl₄solution (1.30 ml, 1.0 M solution in CH₂ Cl₂, 1.30 mmol) was added,dropwise, at room temperature. It took 2 hours to complete the reaction.Upon completion, the solution was concentrated, the residue wastriturated with pyridine (2 ml) and subjected to flash chromatography(first with neat EtOAc then 20% EtOH in EtOAc) to give a tan solid,which was further recrystallized to give a white crystalline solid 32(0.25 g, 0.55 mmol, 89%). (¹ H NMR (CDCl₃, 300 MHz) 7.97 (1H, d, H_(1'),J=7.8 Hz), 7.67 & 7.40 (10H, m, aromatic-H), 6.24 (1H, d, H_(1')), 5.62(1H, d, H₅, J=7.6 Hz), 5.03 (1H, t, H_(4')), 4.20 (1H, dd, 1H_(2'),J=1.2 and 9.0 Hz), 4.15 (1H, dd, 1H_(2'), J=4.8 & 9.0 Hz), 3.96 (1H, dd,1H_(5'), J=2.1 and 8.7 Hz), 3.93 (1H, dd, 1H_(5'), J=2.1 and 8.7 Hz),1.08 (9H, s, t-Bu).)

Silylether 32 (0.12 g, 0.27 mmol) was dissolved in THF (20 ml), and ann-Bu₄ NF solution (0.30 ml, 1.0 M solution in THF, 0.30 mmol) was added,dropwise, at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up with EtOH/pyridine(2 ml/1 ml), and subjected to flash chromatography (first with EtOAc,then 20% EtOH in EtOAc) to afford a white solid, which was furtherrecrystallized from EtOH to give a white crystalline solid 33 (DOC) (55mg, 0.26 mmol, 96%). (¹ H NMR: (DMSO-d⁶, 300 MHz) 7.79 (1H, d, H₆, J=7.5Hz), 7.18 and 7.11 (2H, broad, NHz), 6.16 (1H, dd, H_(1'), J=3.0 & 4.2Hz), 5.70 (1H, d, H₅, J=7.5 Hz), 5.16 (1H, t, OH, J=6.0 Hz), 4.91 (1H,t, H_(4'), J=2.7 Hz), 4.05 (2H, m, H_(2')), 3.62 (2H, m, 2H_(5')); mp183°-184° C.)

The coupling reaction of acetate 29 with silylated thymine 31 showed atitanium species dependent selectivity in accordance with the followingobservations (ratios were determined by ¹ H NMR of the crude reactionmixtures):

    ______________________________________                                        Titanium species    β:α Ratio                                      ______________________________________                                        TiCl.sub.4          7:1                                                       TiCl.sub.3 (OiPr)   10:1                                                      TiCl.sub.2 (OiPr).sub.2                                                                           >98:2                                                     ______________________________________                                    

In the coupling reaction using TiCl₃ (OiPr), the impure acetate 29 fromthe procedure B reduction above (assumed 69% of the mixture, 185.4 mg,0.4653 mmol) was dissolved in 8 ml of dry dichloromethane along with 144mg (1.15 eq) of silylated thymine 31, and this mixture was stirred underargon at room temperature. Next 0.57 ml (1.15 eq) of a freshly preparedsolution of TiCl₃ (OiPr) in dichloromethane (1 M solution prepared from2 eq of TiCl₄ and 1 eq of TiCl(OiPr)₃) was added dropwise over a 25minute period. After 2.5 hours, 0.07 ml (0.15 eq) of a TiCl₄/dichloromethane solution (1 M, Aldrich) was added and the reaction wasstirred for an additional hour. Then 3 ml of ethanol and 5 ml of NaHCO₃solution were added, stirred for 10 minutes, followed by extraction withadditional NaHCO₃ solution. The aqueous layer was separated, washedtwice with 100 ml of dichloromethane, and the organic layers werecombined and dried over MgSO₄. Filtration, solvent removal, columnchromatography (1/2: Hexanes/EtOAc), and then recrystallization (1/1:Hexanes/Et₂ O) gave 160 mg (74%) of compound 34 as a white powder. (¹ HNMR: (CDCl₃, 300 MHz) 1.06 (s, 9H), 1.68 (s, 3H), 3.91 (t, 2H, J= 3.3Hz), 4.14 (d, 2H, J=3.9 Hz), 5.06 (t, 1H, J=3.3 Hz), 6.34 (t, 1H, J=3.9Hz), 7.4 (m, 6H), 7.7 (m, 4H), 8.62 (bs, 1H). MS (FAB, Li⁺): 473 (M+Li),409, 307, 241, 197, 154, 127. Anal. Calc. for C₂₅ H₃₀ O₅ N₂ Si: C,64.35%; H, 6.48%; N, 6.00%; Found: C, 64.42%; H, 6.52%; N, 5.97%.)

In the coupling reaction using TiCl₂ (OiPr)₂, impure acetate from theprocedure B reduction (assumed 50% of the mixture, 444 mg, 1.11 mmol)was dissolved in 18 ml of dry dichloromethane along with 654.1 mg ofsilylated thymine 31 and stirred at room temperature under argon. Next,1.3 ml of a 2 M TiCl₂ (OiPr)₂ /CH₂ Cl₂ solution was added over a 20minute period. After 14 h, 1 ml of a 1 M TiCl₄ /CH₂ Cl₂ solution wasadded and the reaction was stirred for an additional 3 hours. Then 4 mlof concentrated NH₄ OH was added, along with 10 ml of dichloromethane.Ten minutes of stirring followed by filtration over 1 inch of silica gelwith EtOAc, solvent removal and then column chromatography of theresulting oil gave 164.9 mg (32%) of compound 34.

The silyl ether 34 (60.9 mg, 0.131 mmol) was dissolved in 2 ml of THFand 0.14 ml of a Bu₄ NF/THF solution (1 M, Aldrich) was added. Afterstirring for 24 hours, the solvent was removed envaccuo and columnchromatography (5/1: EtOAc/EtOH) of the resulting oil gave 22.6 mg (76%)of the desired nucleoside 35 (DOT) as a white powder. (¹ H NMR: (HOD(4.8 ppm), 300MHz) 1.83 (s, 3H), 3.82 (m, 2H), 4.18 (dd, 1H, J=10.5 Hz,J=6 Hz), 5.06 (s, 1H), 6.33 (d, 1H, J=5.7 Hz), 7.72 (s, 1H).)

The impure acetate 29 from the procedure B reduction above (assumed 80%by ¹ H NMR analysis, 117.6 mg, 0.294 mmol) and 120.8 mg (1.5 eq) ofsilylated fluorocytosine 30 (X=F) were dissolved in 10 ml of drydichloromethane. Then 0.59 ml (2 eq) of a TiCl₄ /dichloromethanesolution was added dropwise over 1 hour. After stirring for 30additional minutes, 5 ml of dichloromethane and 1 ml of concentrated NH₄OH were added, the solvent was removed envaccuo, and columnchromatography (EtOAc/EtOH: 1/1) gave 35 mg (25%) of compound 36 as awhite solid. (¹ H NMR: (CDCl₃, 300 MHz) 1.06 (s, 9H), 3.62 (dq, 2H,J=2.7 Hz, J=12.3 Hz), 3.9 (m, 2H), 5.01 (t, 1H, J=2.4 HZ), 6.2 (m, 1H),7.41 (m, 6H), 7.7 (m, 4H), 7.92 (d, 1H, J=6 Hz).)

The silyl ether 36 (116.8 mg, 0.249 mmol) was dissolved in 3 ml of dryTHF, and 0.3 ml of a Bu₄ NF/THF solution (1M, Aldrich) was added. After3 hours of stirring, the solvent was removed envaccuo and columnchromatography (EtOAc/EtOH: 4/1) gave 48.1 mg (84%) of the nucleoside 37(FDOC) as a white powder. (¹ H NMR: (DMSO-d⁶, 300 MHz) 3.63 (m, 2H),4.01 (dd, 1H, J=5.1 Hz, J=9.6 Hz), 4.08 (d, 1H, J=9.6 Hz), 4.87 (s, 1H),5.26 (t, 1H, J=6 Hz), 6.07 (m, 1H), 7.49 (bs, 1H), 7.73 (bs, 1H), 8.12(d, 1H, J=7.2 Hz).)

B. Therapeutic Use of FTC and FTC Prodrug Analogues

As shown below, the compounds of this invention either possessantiretroviral activity, such as anti-HIV-1, anti-HIV-2 and anti-simianimmunodeficiency virus (anti-SIV) activity, themselves and/or aremetabolizable to species that possess antiretroviral activity. Thus,these compounds, pharmaceutically acceptable derivatives of thesecompounds or pharmaceutically acceptable formulations containing thesecompounds or their derivatives are useful in the prevention andtreatment of viral infections in a host such as a human, preferably HIVinfections and other AIDS-related conditions such as AIDS-relatedcomplex (ARC), persistent generalized lymphadenopathy (PGL),AIDS-related neurological conditions, anti-HIV antibody positive andHIV-positive conditions, Kaposi's sarcoma, thrombocytopenia purpurea andopportunistic infections. In addition, these compounds or formulationscan be used prophylactically to prevent or retard the progression ofclinical illness in individuals who are anti-HIV antibody or HIV-antigenpositive or who have been exposed to HIV.

As used herein, a "pharmaceutically acceptable derivative" means anypharmaceutically acceptable salt, ester, or salt of such ester, of FTCor a prodrug analogue of FTC which, upon administration to therecipient, is capable of providing, directly or indirectly, FTC or anantivirally active metabolite or residue of FTC, including, but notlimited to, the mono-, di- and triphosphate esters of FTC or a prodruganalogue of FTC.

Thus, humans can be treated by administering to the patient apharmaceutically effective amount of FTC or FTC prodrug analogues in thepresence of a pharmaceutically acceptable carrier or diluent such as aliposomal suspension. A preferred carrier for oral administration iswater, especially sterilized water. If administered intravenously, thepreferred carriers are physiological saline or phosphate bufferedsaline. The compounds according to the present invention are included inthe pharmaceutically acceptable carrier in an amount sufficient to exerta therapeutically useful inhibitory effect on HIV in vivo withoutexhibiting adverse toxic effects on the patient treated.Pharmaceutically compatible binding agents and/or adjuvant materials mayalso be included as part of the composition. The active materials canalso be mixed with other active materials that do not impair the desiredaction and/or supplement the desired action.

It will be appreciated by those skilled in the art that the effectiveamount of a compound or formulation containing the compound required totreat an individual will vary depending on a number of factors,including whether FTC or a prodrug analogue of FTC is administered, theroute of administration, the nature of the condition being treated andthe age and condition of the patient. In general, however, an effectivedose will range from about 1-50 mg per kg body weight of the patient perday, preferably 1-20 mg/kg/day. Preferably, a dose will produce peakblood levels of the active compound that range from about 1-10 μM, mostpreferably about 5 μM. The desired dose may be given in a single dose oras divided doses administered at appropriate intervals, such as two,three, four or more sub-doses per day.

Thus, FTC and FTC prodrug analogues or formulations containing thesecompounds or their pharmaceutically acceptable derivatives can beconveniently administered by any convenient route of administration,such as parenteral, including intramuscular, subcutaneous andintravenous; oral; rectal; nasal; vaginal or by inhalation. Thecompounds can be administered in unit dosage form, such as formulationscontaining 0.1 to 50 mg, preferably, 1 to 10 mg of active ingredient perunit dosage form.

A preferred mode of administration of the compounds of this invention isoral. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, thecompounds of this invention may be incorporated with excipients and usedin the form of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

Methodology for Testing Antiviral Activity

Antiviral compositions can be screened in vitro for inhibition of HIV byvarious experimental techniques. One such technique involves measuringthe inhibition of viral replication in human peripheral bloodmononuclear (PBM) cells. The amount of virus produced is determined bymeasuring the quantity of virus-coded reverse transcriptase (RT), anenzyme found in retroviruses, that is present in the cell culturemedium.

PBM cells from healthy HIV-1 and hepatitis B virus seronegative donorswere isolated by Ficoll-Hypaque discontinuous gradient centrifugation at1,000×g for 30 minutes, washed twice in PBS and pelleted at 300×g for 10minutes. Before infection, the cells were stimulated byphytohemagglutinin (PHA) at a concentration of 6 μg/ml for three days inRPMI 1640 medium supplemented with 15% heat-inactivated fetal calfserum, 1.5 mM L-glutamine, penicillin (100 U/ml), streptomycin (100μg/ml), and sodium bicarbonate buffer. Most of the antiviral assaysdescribed below were performed with cells from at least two differentdonors.

HIV-1 (strain LAV-1) was obtained from the Centers for Disease Control,Atlanta, and propagated in PHA-stimulated human PBM cells using RPMI1640 medium as above without PHA and supplemented with 7% interleukin-2(Advanced Biotechnologies, Silver Spring, Md.), 7 μg/ml DEAE-dextran(Pharmacia, Uppsala, Sweden), and 370 U/ml anti-human leukocyte (alpha)interferon (ICN, Lisle, Ill.). Virus was obtained from the cell freeculture supernatant and stored in aliquots at -70° C. until used.

Uninfected PHA-stimulated human PBM cells were uniformly distributedamong 25 cm³ flasks to give a 5 ml suspension containing about 2×10⁶cells/ml. Suitable dilutions of HIV were added to infect the cultures sothat the mean reverse transcriptase (RT) activity of the inocula was50,000 dpm/ml, which was equivalent to about 100 TCID50, determined asdescribed in AIDS Res. Human Retro, 3:71-85 (1987). The drugs, at twicetheir final concentrations in 5 ml of RPMI 1640 medium, supplemented asdescribed above, were added to the cultures. Uninfected and treated PBMcells were grown in parallel as controls. The cultures were maintainedin a humidified 5% CO₂ -95% air incubators at 37° C. for five days afterinfection, at which point all cultures were sampled for supernatant RTactivity. Previous studies indicate that the maximum RT levels areobtained at that time.

The RT assay was performed by a modification of the Spira et al., J.Clin. Microbiol. 25, 97-99 (1987) method in 96-well microtiter plates.The radioactive cocktail (180 μl), which contained 50 mM Tris-HCl pH7.8, 9 mM MgCl₂, 5 mM dithiothreitol 4.7 μg/ml (rA)n.(dT)12-18, 140 μMdATP and 0.22 μM [³ H]TTP (specific activity 78.0 Ci/mmol, equivalent to17,300 cpm/pmol; NEN Research Products, Boston, Mass.), was added toeach well. The sample (20 μl) was added to the reaction mixture andincubated at 37° C. for two hours. The reaction was terminated by theaddition of 100 μl cold 10% trichloroacetic acid (TCA) containing 0.45mM sodium pyrophosphate. The acid insoluble nucleic acid whichprecipitated was collected on glass filters using a Skatronsemiautomatic harvester (setting 9). The filters were washed with 5% TCAand 70% ethanol, dried, and placed in scintillation vials. Four ml ofscintillation fluid (Econofluor, NEN Research Products, Boston Mass.)was added and the amount of radioactivity in each sample determinedusing a Packard Tri-Carb liquid scintillation analyzer (model 2,000CA).The results were expressed in dpm/ml of original clarified supernatant.The antiviral activity, expressed as the micromolar concentration ofcompound that inhibits replication of the virus by 50% (EC₅₀), wascalculated by determining the percent inhibition by the median effectmethod described in Chou and Talalay, Adv. Enz. Regul., 22:27-55 (1984).

Methodology for Testing Toxicity and Inhibition of Cell Proliferation

The compounds were evaluated for their potential toxic effects onuninfected PHA-stimulated human PBM cells and also in CEM(T-lymphoblastoid cell line obtained from ATCC, Rockville, MD) and Vero(African Green Monkey kidney) cells. PBM cells were obtained from wholeblood of healthy HIV and hepatitis-B seronegative volunteers andcollected by a single-step Ficoll-Hypaque discontinuous gradientcentrifugation. The CEM cells were maintained in RPMI 1640 mediumsupplemented with 20% heat-inactivated fetal calf serum, penicillin (100U/ml), and streptomycin (100 μg/ml). Flasks were seeded so that thefinal cell concentration was 3×10⁵ cells/ml. The PBM and CEM cells werecultured with and without drug for 6 days at which time aliquots werecounted for cell proliferation and viability using the trypanblue-exclusion method (Sommadossi et al, Antimicrob. Agents Chemother.,32:997-1001 (1988). Only the effects on cell growth are reported becausethese correlated well with cell viability. The toxicity of the compoundsin Vero cells was assessed after 3 days of treatment with ahemacytometer as described in Schinazi et al, Antimicrob. AgentsChemother., 22:499-507 (1982). The toxicity, expressed as the micromolarconcentration of compound that inhibits the growth of normal cells by50% (IC₅₀), was determined, similarly to EC₅₀, by the method of Chou andTalalay.

In Vitro Assay is Predictive of In Vivo Activity

Using the antiviral activity PBM assay described above, a number ofcompounds have been tested for activity against HIV. While many of thecompounds have been found to have little or no activity against thevirus under the test conditions, a number of the compounds haveexhibited significant activity. For instance, DDI, DDC, D4T, AzddU(3'-Azido-2',3'-dideoxyuridine) and AZT were found to significantlyinhibit HIV replication in vitro. and to have low cytotoxicity in PBMcells under the test conditions used. FTC also exhibits significantactivity against HIV replication in the PBM cell line assay.

At least four of the compounds found active in the PBM cell line assay(DDI, DDC, DDA, and AzddU) are undergoing clinical testing in the U.S.Food and Drug Administration (FDA). All four compounds have been foundto inhibit HIV in vivo. A fifth compound, AZT, is already approved bythe FDA for treatment of HIV in humans. Based on the correlation of theresults of the in vitro PBM assay with in vitro activity, it is clearthat the activity of a compound against HIV in the PBM cell line invitro is fairly predictive of its general activity in vivo in humans.

EXAMPLE 7--ANTIVIRAL AND CYTOTOXICITY ASSAYS OF FTC AND3'-THIANUCLEOSIDE ANALOGUES OF FTC IN HUMAN PERIPHERAL BLOOD MONONUCLEAR(PBM) CELLS.

Table 1 below lists the results of anti-HIV-1 activity and toxicityassays in human PBM Cells as described above for various3'-thianucleoside analogues related to BCH-189. It appears that only thecytidine analogues are active in PBM cells, especially when the5-position is substituted with H or F; FTC was more potent an inhibitorthan any of the other tested compounds. Surprisingly, the 5-methylderivative was inactive when tested up to 100 μM. These compounds werenot cytotoxic to human PBM cells when tested up to 100 μM. Cells from atleast two different donors were used in performing these antiviralassays. The margin of inter-assay variability error in EC₅₀ valuesdetermined from a concentration-response curve can vary by as much as afactor of 10. However, using the above procedure and AZT as a positivecontrol, a variance of 0.0008 to 0.006 μM with a mean value of 0.002 μMwas determined.

                  TABLE 1                                                         ______________________________________                                        Anti-HIV Activity and Toxicity of Various                                     Analogues of 2'-deoxy-3'-thiacytidine                                         in Human PBM Cells                                                            Antiviral Drug        EC.sub.50,μM                                                                        IC.sub.50,μM                                ______________________________________                                        2',3'-Dideoxy-3'-thiauridine                                                                        >100     >100                                           2'-Deoxy-5-methyl-3'-thiauridine                                                                    64.4     >100                                           2'-Deoxy-5-fluoro-3'-thiauridine                                                                    >100     >100                                           2'-Deoxy-5-chloro-3'-thiauridine                                                                    >60.8    >100                                           2'-Deoxy-5-bromo-3'-thiauridine                                                                     NA       NA                                             2'-Deoxy-5-iodo-3'-thiauridine                                                                      >100     >100                                           2'-Deoxy-3'-thiacytidine (BCH-189)                                                                  0.05     >100                                           2'-Deoxy-5-methyl-3'-thiacytidine                                                                   10       >100                                           2'-Deoxy-5-fluoro-3'-thiacytidine (FTC)                                                             0.011    >100                                           2'-Deoxy-5-chloro-3'-thiacytidine                                                                   37.8     >100                                           2'-Deoxy-5-bromo-3'-thiacytidine                                                                    7.4      >100                                           2'-Deoxy-5-iodo-3'-thiacytidine                                                                     0.72     >100                                           ______________________________________                                    

Furthermore, as shown in FIG. 6, FTC was highly effective in PBM cellseven when the drug was added 3 days after virus infection. FIG. 6 showsa comparison of the effect of delaying treatment for up to three days onthe anti-HIV-1 activity for FTC, BCH-189, AZT and AzddU. These resultswere determined by measuring the RT activity associated with virionproduced in the presence and absence of drug to quantitate virus yieldas described above. The control for this experiment had 232,154 dpm/mlof RT activity.

It is possible that BCH-189 analogues can be deaminated intracellularlyto the inactive uracil analogue. Close to 6% of BCH-189 can bedeaminated by Cyd/dCyd deaminase in a cell free system. However, thepresence of fluorine in FTC would increase the lipophilicity of thedrug, which should also increase its penetration into the CNS. Inaddition, FTC should be markedly less susceptible to deamination.Deamination of either BCH-189 or FTC would lead to the correspondinguracil analogues, which would cause them to lose their potent activity.

EXAMPLE 8--ANTIVIRAL AND CYTOTOXICITY ASSAYS OF FTC AND AZT IN HUMAN CEMCELLS

FTC was evaluated in vitro versus HIV-1, strain HTLV-III_(B) in CEMcells, a T-cell line, using AZT as the positive control. FTC wasinitially dissolved in sterile water at a concentration of 4 mM, anddilutions were prepared in RPMI-1640 medium containing 10% fetal bovineserum. The compound was tested at nine concentrations, ranging from 100μM to 0.01 μM in half-log₁₀ dilutions.

The assay was done in 96-well tissue culture plates using the CEM humanT-lymphocyte cell line. CEM cells were treated with polybrene at aconcentration of 2 μg/ml, and 1×10⁴ cells were dispensed into each well.A 50 μl volume of each test article dilution, prepared as a 4×concentration, was added to 5 wells of cells, and the cells wereincubated at 37° C. for 1 hour. A frozen culture of HIV-1, strainHTLV-III_(B), was diluted in culture medium and 2×10³ TCID₅₀ of viruswere added to 3 of the wells for each test article concentration. Thisresulted in a multiplicity of infection of 0.2 for the HIV-1 infectedsamples. Normal culture medium was added to the remaining 2 wells ofeach test concentration to allow evaluation of cytotoxicity. Each assayplate contained 2 wells of untreated, uninfected, cell control samplesand 3 wells of untreated, infected, virus control samples. The totalvolume in each well was 200 μl.

Assay plates were incubated at 37° C. in a humidified, 5% CO₂ atmosphereand observed microscopically for toxicity and/or cytopathogenic effect.On the 8th day post-infection, the cells in each well were resuspendedand a 50 μl sample of each cell suspension was transferred to a new96-well plate. A 100 μl volume of fresh RPMI-1640 medium and a 30 μlvolume of a 5 mg/ml solution of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasadded to each 50 μl cell suspension, and the cells were incubated at 37°C. for 4 hours. During this incubation, MTT is metabolically reduced byliving cells, resulting in the production of a colored formazan product.A 50 μl volume of a solution of 20% sodium dodecyl sulfate in 0.02 Nhydrochloric acid was added to each sample, and the samples wereincubated overnight. The absorbance at 590 nm was determined for eachsample using a Molecular Devices V_(max) microplate reader. This assaydetects drug-induced suppression of viral CPE, as well as drugcytotoxicity, by measuring the generation of MTT-formazan by survivingcells.

No cytotoxicity was noted for FTC from 0.01 to 100 μM and the EC₅₀ wasestimated to be 0.09 μM, giving a therapeutic index (IC₅₀ /EC₅₀) inthese cells of about 1000. In contrast, the EC₅₀ for AZT in CEM cellswas 0.01 μM and no cytotoxicity was noted up to 5 μM, the maximumconcentration tested.

EXAMPLE 9--EFFECT OF FTC, BCH-189, AZT AND DDC ON COLONY FORMATION OFGRANULOCYTE-MACROPHAGE PRECURSOR CELLS

Because the limiting toxicity of compounds like AZT is bone-marrowtoxicity, it was important to determine if FTC was also toxic to thesecells. The results of a bone-marrow toxicity assay may predict if anemiawill occur in humans following treatment with a particular drug becausethese cell culture models are good prognosticators of what may happen inhumans. Thus, FTC, BCH-189, DDC and AZT were tested for their effects oncolony formation of granulocyte-macrophage precursor cells.

Human bone marrow cells were collected by aspiration from the posterioriliac crest of normal healthy volunteers, treated with heparin and themononuclear population separated by Ficoll-Hypaque gradientcentrifugation. Cells were washed twice in Hanks balanced salt solution,counted with a hemacytometer, and their viability was >98% as assessedby trypan blue exclusion. The culture assays were performed using abilayer soft-agar or methyl cellulose method. McCoy 5A nutrient mediumsupplemented with 15% dialyzed fetal bovine serum (heat inactivated at56° C. for 30 minutes, Gibco Laboratories, Grand Island, N.Y.) was usedin all experiments. This medium was devoid of thymidine and uridine.Human recombinant GM-CSF (50 units/ml, Genzyme, Boston, Mass.) was usedas colony-stimulating factors. After 14 days of incubation at 37° C. ina humidified atmosphere of 5% CO₂ in air, colonies (>50 cells) werecounted using an inverted microscope.

As shown in FIG. 7, studies with human bone marrow cells indicate thatFTC has an IC₅₀ greater than 50 μM, whereas in the same assay BCH-189,DDC, and AZT are clearly more toxic. The IC₅₀ for AZT is close to 1 μM.

Because both BCH-189, AZT and FTC do not seem to affect theproliferation of uninfected human PBM cells as shown above, it isimportant to calculate the therapeutic index of the drugs in terms ofIC₅₀ (toxicity) in human bone-marrow cells to EC₅₀ (antiviral) againstHIV in human PBM cells. The IC₅₀ in human bone-marrow cells for BCH-189is about 10 μM, whereas for FTC it is about 60 μM. Hence the therapeuticindex for BCH-189 is 10/0.05=200, while the index for FTC is60/0.011=5,455. By these experiments, FTC is clearly a less toxic yeteffective anti-HIV-1 agent compared to BCH-189.

EXAMPLE 10--ANTVIRAL AND CYTOTOXICITY ASSAYS OF FTC in MT-2 CELLS

Antiviral and cytotoxicity studies of FTC in human lymphocyte MT-2 cellswere conducted. MT-2 cells (3×10⁵ /ml) were incubated with serial10-fold dilutions of an HIV (IIIb) viral supernatant (stock),centrifuged, resuspended in fresh media, and plated into microculturewells (6×10⁴ cell/well/0.2 ml). Because the assay can be performed with0.2 ml of culture supernatant in a microtiter plate, HIV inoculation oftarget cell cultures can be monitored conveniently and endpointtitrations of infectious HIV can be performed. No manipulation of theculture is required during the seven day evaluation. The necessarymultiple replicate numbers of cultures to generate statisticallysignificant data were included in the TCID₅₀ assay. Since the MT-2 cellline is highly susceptible to virus infection and syncytia formation, itis easily observed and allows for a very sensitive assay system.

Quantitation of HIV infectivity was determined for serial 10-folddilutions of the virus stock. Calculation of the highest dilution ofvirus which gave evidence of syncytia in 50% of the cultures, theendpoint determination, yielded a measure of the infectious particles inthe stock. A TCID₅₀ titer is defined as the reciprocal of the dilutionof HIV that when inoculated into the microcultures containing MT-2 cellsresulted in syncytia in 50% of the cultures by the seventh day. Theresults of the HIV TCID₅₀ assay, as described in Table 2, correlateswith the results using the reverse transcriptase results,immunofluorescent, cytoplasmic staining assay, p24 antigen captureassay, and cell cytopathic effects, thereby validating our assay system.

The MT-2 syncytium-forming assay has been applied for use in discoveringantiviral drugs with potent anti-HIV activity. MT-2 cells are incubatedin growth medium (DMEM, 20% heat inactivated fetal calf serum and 0.25mg/ml L-glutamine with 1% penicillin and streptomycin) at 37° C. in a 5%CO₂ atmosphere. The MT-₂ cell concentration that allows for thedevelopment of readily quantifiable syncytium formation in a microtiterplate is 3×10⁵ /ml (6×10⁴ cell/0.2 ml).

HIV (IIIb) was obtained from the culture supernatant of H9 cellsinfected by multiple isolates of HIV concentrated to 10,000× by sucrosegradient centrifugation. A representative virus (IIIb) stock contained atotal virus particle count of approximately 10⁸ /ml to 10⁹ /ml byelectron microscopy. The TCID₅₀ was calculated as follows: Serial10-fold dilutions of the H9 virus stock were performed and 1.0 ml used(in quadruplicate) to infect MT-2 cells. Endpoints were calculated bythe method of Reed Muench from the highest dilution with detectablesyncytium formation within seven days. The most recent virus stock, HIV(IIIb), that was evaluated contained an infectious viral titer of 6.23log₁₀ TCID₅₀ /ml. The input dose of virus was adjusted to yield greaterthan 40 syncytia at the seventh day of culture. HIV stocks werealiquoted and stored at -85° C. until used. A frozen stock was thawedand an infectivity study was performed, in quadruplicate, to determineif >40 syncytia are formed at day seven. At the same time, the virusstock was subjected to antiviral inhibition with the use of AZT or DDA.These maneuvers, with the proper controls, ensure for reproducible inputdoses of virus for these studies.

                  TABLE 2                                                         ______________________________________                                        EFFECT OF BCH-189 AND FTC AGAINST HIV-1                                       (strain IIIb) IN MT-2 CELLS                                                                    Mean # of                                                             Conc.   Syncytia    %                                                Compound (μM) (per well)  Inhib EC.sub.50,μM                            ______________________________________                                        Cells (no        0           0.00                                             virus/no                                                                      drug                                                                          Virus (no        62          0.00                                             drug)                                                                         DDA (pos.                                                                              1       14          77.42 ≈0.45                              control  10      0.5         99.19                                            BCH-189  0.1     61          1.61  0.88                                                1       20.5        66.94                                                     10      1           93.39                                                     100     0           100.0                                            FTC      0.1     63          -1.61 0.89                                                1       23          62.90                                                     10      0.5         99.19                                                     100     0           100.00                                           ______________________________________                                    

The MT-2 cells for the studies were expanded and treated withDEAE-dextran (25 μg/ml) for 20 minutes followed by three washings withPBS. Cell counts were performed and an appropriate number of cells thatultimately yielded a final cell concentration of 3×10⁵ cells/ml (6×10⁴cells/0.2 ml) per well was chosen. The cells were infected in bulk (notin-well infection) at a multiplicity of infection of 10⁻³ and allowed tomix with the viral supernatant for one hour at 37° C. The cells weresubsequently resuspended in the wells containing the MT-2 media withdrugs. The cultures were not manipulated until day seven when syncytiumcounts and cell viability studies were performed. The experimentalcontrols for each experiment consisted of the following: 1) AZT or DDA;2) uninfected MT-2 cells with drug; 3) infected MT-2 cells without drug,and 4) uninfected MT-2 cells without HIV or drugs.

The raw data was analyzed by the method of Chou and Talalay. The MT-2cell lines were discarded at three month intervals with a new stockregrown to avoid the possibility of variations or contamination(mycoplasma) with long term growth. The original MT-2 cell frozen stockhas been tested and is free of mycoplasma. EC₅₀ and IC₅₀ values wereobtained by analysis of the data using the median-effect equation ofChou and Talalay. It is apparent from Table 2 that in this cell culturesystem, both BCH-189 and FTC are equally potent.

EXAMPLE 11--INHIBITION OF MITOCHONDRIAL DNA SYNTHESIS BY FTC IN CEMCELLS

In addition to bone-marrow toxicity, peripheral neuropathy has beenobserved with certain nucleoside antiviral drugs. There appears to be agood correlation between inhibition by nucleosides of mitochondrial DNAsynthesis and clinical peripheral neuropathy. Therefore, studies wereperformed which indicated that FTC did not affect mitochondrial DNAsynthesis in intact CEM cells when tested up to 100 μM. This result wasdetermined by measuring the amount of mitochondrial DNA present in theselymphocytes after exposure using a mitochondrial DNA hybridizationprobe. However, BCH-189 and DDC are toxic in this system at aconcentration ≦10 μM.

EXAMPLE 12--EFFECT OF FTC, BCH-189 AND AZT ON AZT-RESISTANT ANDAZT-SENSITIVE HIV-1 IN HUMAN PBM CELLS

We have also evaluated FTC and BCH-189 against AZT-resistant andsensitive HIV-1, as shown in FIG. 8 and Table 3. The pairedAZT-resistant and sensitive viruses strain 9F (G910-6) and 10 (HI12-2),respectively, were obtained through the NIH AIDS Research and ReferenceProgram. All the viruses were propagated in PHA-stimulated human PBMcells using RPMI 1640 medium as described previously and supplementedwith 7% interleukin-2 (Advanced Biotechnologies, Silver Spring, Md.), 7μg/ml DEAE-dextran (Pharmacia, Uppsala, Sweden), and 370 U/ml anti-humanleucocyte (alpha) interferon (ICN, Lisle, Ill.). Virus was obtained fromcell-free culture supernatant and stored in aliquots at -70° C. untiluse. The antiviral assay in PBM cells was performed as described above.

                  TABLE 3                                                         ______________________________________                                                  EC.sub.50,μM                                                     Compound  Strain 9F*   Strain 10                                                                              Fold Increase                                 ______________________________________                                        AZT       0.298        0.00069  432                                           BCH-189   0.244        0.040    6.1                                           FTC       0.107        0.014    7.6                                           ______________________________________                                    

At the same multiplicity of infection, a 7-fold increased resistance wasnoted at the EC₅₀ level when the sensitivity of the pretherapy isolatewas compared to the post-therapy AZT-resistant virus in PBM cells forFTC. This increase was not as great as that noted for AZT.

EXAMPLE 13--INHIBITORY EFFECT OF FTC AGAINST SIV₂₅₁

FTC was tested for its inhibitory effect against SIV₂₅₁ in the humancell line AA-2 and C-8166, using AZT as a positive control. All testswere conducted in duplicate according to a standard protocol in 96 welltissue culture plates. Briefly, cells were exposed to the virus for 1hour at 37° C. The cells were washed and the appropriate dilution ofantiretroviral agent diluted in PBS was added with complete RPMI-1640medium. After a 7-day incubation period at 37° C. and 5% CO₂, 95% airenvironment, cells were examined microscopically for cytopathic effects(syncytial cells) and cytotoxicity. The cells were counted and thepercent of viable cells determined using the trypan blue exclusionmethod. Viral antigen expression in cell pellets was determined by animmunofluorescence (IF) assay. The percent of IF inhibition was based onthe ratio of fluorescing cells in infected/treated cultures tofluorescing cells in infected control cultures.

FTC antiviral activity was observed versus SIV but less than that notedwith AZT. As shown in Table 4, FTC was evaluated over a concentrationrange of 0 to 46 μM, and AZT was tested as the positive control.

                  TABLE 4                                                         ______________________________________                                        Concentration                                                                             % IF Inhibition   Cell No. × 10.sup.5                       (μM)     AA-2    C-8166    AA-2  C-8166                                    ______________________________________                                        FTC  0          0       0       6.0   3.9                                          0.23       0       11      6.5   5.6                                          0.46       17      5       6.5   6.0                                          2.3        22      32      6.3   5.9                                          4.6        36      47      7.3   6.1                                          23         61      63      6.2   7.4                                          46         70      79      9.9   9.9                                     AZT  0.0005     0       5       7.4   6.6                                          0.005      30      16      7.4   8                                            0.05       83      79      7.5   8                                            0.5        100     100                                                   ______________________________________                                    

EXAMPLE 14--THYMIDYLATE SYNTHASE ASSAY OF FTC AND BCH-189

BCH-189 and FTC were also evaluated in an intact L1210 cellularthymidylate synthase (TS) assay. No evidence for any inhibition of TS byup to 1 mM of either compound as measured by the release of tritium from5-³ H-dUrd was noted. Using 5-³ H-dCyd, inhibition of tritium releasewas observed at >10⁻⁴ M. At 1 mM, BCH-189 and FTC gave 63.2% and 74.7%inhibition of tritium release, respectively. Since the 5-³ H-dCydconcentration is 1 μM, it appears that the observed effects may be dueto competitive inhibition of the phosphorylation of labeled dCyd by theanalogue at high concentrations. The lack of TS inhibition by FTC isprobably due to either of 2 alternatives: (1) its 5'-phosphate is not asubstrate for dCMP deaminase; (2) if it is a substrate, the resulting5-fluoro-3'-thia-dUMP cannot bind to TS or, if so, only very weakly.

EXAMPLE 15--ANTIVIRAL ACTIVITY OF VARIOUS PRODRUGS OF FTC IN HUMAN PBMCELLS

FTC may be modified at the 2-hydroxymethyl group of the oxathiolane ringby substituting the hydroxy group with an oxyacyl group to produce5'-oxyacyl or 5'-H substituted prodrug analogues of FTC. Furthermore,the 4-N position of FTC may be substituted with an alkyl, substitutedalkyl, cycloalkyl or acyl group. These modifications at the 4-N and 5'-Opositions affect the bioavailability and rate of metabolism of theactive species, thus providing control over the delivery of the activespecies.

Preferred FTC prodrug analogues include compounds of the formula:##STR7## in which Y₁ and Y₂ are selected from H; lower straight orbranched chain alkyl; substituted alkyl, preferablydiisopropylaminomethylene or alkoxyaminomethylene; cycloalkyl,preferably cyclopropyl; or acyl, wherein the term "acyl" corresponds toan acyl protecting group as given above and in which the 5'-Rsubstituent is H or oxyacyl. As used herein, the term "oxyacyl" means agroup of the formula ##STR8## in which R' is selected from hydrogen,lower straight or branched- chain alkyl (e.g., methyl, ethyl, n-propyl,t-butyl, n-butyl), alkoxyalkyl (e.g., methoxymethyl), aralkyl (e.g.,benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyl),substituted aryl (e.g., halogen, lower alkyl or lower alkoxy substitutedphenyl); substituted dihydro pyridinyl (e.g., N-methyldihydropyridinyl); sulphonate esters such as alkyl- or aralkylsulphonyl (e.g.,methanesulphonyl); sulfate esters; amino acid esters (e.g., L-valyl orL-isoleucyl) and mono-, di- or tri-phosphate esters. Pharmaceuticallyaccepted formulations of these compounds include liposome formulations.

                  TABLE 5                                                         ______________________________________                                        NY.sub.1 Y.sub.2                                                                      5-position 5'-position   EC.sub.50,μM                              ______________________________________                                        NHAc    H          CH.sub.2 OH   0.089                                        NH.sub.2                                                                              H          n-C.sub.3 H.sub.7 C(O)OCH.sub.2                                                             0.037                                        NH.sub.2                                                                              H          CH.sub.3 C(O)OCH.sub.2                                                                      0.089                                        NHAc    H          n-C.sub.3 H.sub.7 C(O)OCH.sub.2                                                             0.11                                         NHAc    F          n-C.sub.3 H.sub.7 C(O)OCH.sub.2                                                             0.00576                                      NHAc    F          CH.sub.2 OH   0.0028                                       NH.sub.2                                                                              F          n-C.sub.3 H.sub.7 C(O)OCH.sub.2                                                             0.00174                                      ______________________________________                                    

Using the method of determining anti-HIV-1 activity as described inExample 6 above, various prodrugs of FTC and BCH-189 were assayed inhuman PBM cells infected with HIV-1, as shown in Table 5. Relative tothe BCH-189 prodrug analogues listed in Table 5, the FTC prodruganalogues showed superior anti-HIV activity.

EXAMPLE 16--ANTIVIRAL AND CYTOTOXICITY ASSAYS OF NUCLEOSIDES SIMILAR TOFTC

Table 6 below lists the results of anti-HIV-1 activity in human PBMcells and toxicity assays in human PBM cells, Vero (African Green Monkeykidney) cells, and CEM cells as described above for FTC, BCH-189,2'-deoxy-3'-oxacytidine (DOC), 2'-deoxy-3'-oxathymidine (DOT),2'-deoxy-5-fluoro-3'-oxacytidine (FDOC) and2'-deoxy-5-fluoro-3'-oxauridine (FDOU) to show the effect of fluorosubstitution at the 5-position and S→O substitution at the 3'-positionin nucleosides that are similar to FTC.

Comparison of the data for FTC, FDOC and FDOU shows that 5-fluorosubstitution leads to unpredictable results in these systems. Forinstance, fluoro substitution of BCH-189 at the 5-position to give FTCresults in a compound that possesses better anti-HIV activity and isless toxic in CEM cells; both are nontoxic in PBM and Vero cells.However, fluoro substitution of DOC at the 5-position to give FDOCresults in a compound that possesses inferior anti-HIV activity and ismore toxic in Vero cells; both are nontoxic in PBM and toxic in CEMcells. FDOU is nontoxic in all three types of cells but does not possessanti-HIV activity.

Similarly, comparison of the data for FTC, BCH-189 versus DOC, DOT, FDOCand FDOU shows that 3'-substitution of an S for an O gives rise tounpredictable anti-HIV activity and toxicity behavior. For instance,substitution of BCH-189 to give DOC and FTC to give FDOC results incompounds that are toxic in the rapidly dividing Vero cells and CEMcells, thus most likely rendering them not viable as anti-HIV drugsbecause of associated side effects. However, the presence of the oxygenat the 3'-position in DOT does not render this compound toxic in Verocells. Thus, discovery of the superior anti-HIV and toxicity propertiesof FTC was surprising and unexpected.

                  TABLE 6                                                         ______________________________________                                        Anti-HIV Activity and Toxicity of Various                                     Nucleosides that are similar to FTC                                           ANTI-HIV                                                                      ACTIVITY        CYTOTOXICITY                                                  Antiviral                                                                             EC.sub.50,μM                                                                           IC.sub.50,μM                                                                         IC.sub.50,μM                                                                      IC.sub.50,μM                          Drug    (PBM)       (PBM)     (Vero) (CEM)                                    ______________________________________                                        FTC     0.011       >100      >100   >100                                     BCH-189 0.06        >100      >100   52.6                                     DOC     0.0047      >200      0.17   <1                                       DOT     0.09        >100      >100                                            FDOC    0.0063      >200      <0.1   <1                                       FDOU    >10         >200      >100   >100                                     ______________________________________                                    

EXAMPLE 17--EFFECT OF FTC AND BCH-189 ON MITOGENIC STIMULATION

Peripheral blood mononuclear cells (PBM cells) were obtained byleukophoresis from a normal human donor and were further purified bydensity gradient centrifugation using Histopaque (Sigma; St. Louis,Mo.). Cells were washed twice in phosphate buffered saline, resuspendedin complete media (RPMI supplemented with 10% fetal bovine serum, 2 μML-glutamine, penicillin, and streptomycin), and adjusted to 2×10⁶cells/ml. Mitogens were added to separate aliquots of cell suspension toyield a final concentration of 1% phytohemagglutinin (PHA, a T-helpercell mitogen), 0.8 mg/ml concanavalin A (con A, a T-cytotoxic/suppressorcell mitogen), and 0.1% pokeweed mitogen (PWM, a B cell mitogen),respectively.

A cell suspension (100 μl) was dispensed into wells of 6-wellflat-bottomed plates, followed by addition of 100 μl of drug diluted incomplete media. Control wells received 100 μl of complete media. Cellswere incubated at 37° C. in 5% CO₂ for 54 hr, at which time 2 μCi ³H-deoxyguanosine (Moravek Biochemicals, Brea, Calif.; diluted in 20 μlcomplete media) was added per well. After an additional 18 hourincubation, cells were harvested on filter paper using a Skatron cellharvester with 5% TCA and 70% ETOH. Filters were placed in scintillationvials with 4 ml Ecolite, and dpm were counted using a Beckman LS3801beta counter.

At concentrations of 0.1, 1.0, and 10 μM, both BCH-189 and FTC increasedthe proliferation of PBM cells exposed to PHA, whereas they causedsignificant reduction in proliferation at 100 μM concentrations. Con A-and PWM-stimulated cells were suppressed by both drugs. In the absenceof mitogen, BCH-189 has a mildly stimulatory effect, whereas FTC had amildly inhibitory effect.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description of theinvention. Such modifications and variations are intended to come withinthe scope of the appended claims. The references cited above are herebyincorporated by reference to more fully describe the invention.

What is claimed is:
 1. A method for treating a human having an HIVinfection comprising administering to the human an effectiveHIV-treatment amount of a compound of the formula: ##STR9##
 2. Themethod of claim 1, wherein the pharmaceutically acceptable derivativecomprises a monophosphate, diphosphate, or triphosphate ester of2'-deoxy-5-fluoro-3'-thiacytidine.
 3. The method of claim 1, wherein thepharmaceutically acceptable carrier comprises a liposomal suspension. 4.A method for treating a human having an HIV infection comprisingadministering to the human an effective HIV-treatment amount of aβ-isomer of a substituted 2'-deoxy-5-fluoro-3'-thiacytidine having theformula: ##STR10## wherein Y is independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl and acyl; andR isselected from the group consisting of H, hydroxyl, oxyacyl,monophosphate, diphosphate, and triphosphate.
 5. The method of claim 4,wherein the substituted 2'-deoxy-5-fluoro-3'-thiacytidine is selectedfrom the group consisting of the monophosphate, diphosphate, andtriphosphate ester of 2'-deoxy-5-fluoro-3'-thiacytidine.
 6. The methodof claim 4, wherein the pharmaceutically acceptable carrier comprises aliposomal suspension.
 7. The method of claim 4, wherein the β-isomer ofthe substituted 2'-deoxy-5-fluoro-3'-thiacytidine is selected from thegroup consisting of 4-N-acetyl-2'-deoxy-5-fluoro-3'-thiacytidine;4-N-acetyl-5'-butyryl-2'-deoxy-5-fluoro-3'-thiacytidine; and5'-butyryl-2'-deoxy-5-fluoro-3'-thiacytidine.
 8. A method for treating ahuman having an HIV infection comprising administering to the human aneffective HIV treatment amount of a compound of the formula: ##STR11##wherein Y is monophosphate, diphosphate, or triphosphate.
 9. A methodfor treating a human having an HIV infection comprising administering tothe human an effective HIV-treatment amount of a compound of theformula: ##STR12## wherein Y is independently selected from the groupconsisting of H, alkyl, cycloalkyl, and acyl; andR is selected from thegroup consisting of H, hydroxyl, oxyacyl, monophosphate, diphosphate,and triphosphate.
 10. A method for treating a human having an HIVinfection comprising administering to the human an effectiveHIV-treatment amount of a pharmaceutically acceptable salt of a compoundof the formula: ##STR13##
 11. A method for treating a human having anHIV infection comprising administering to the human an effectiveHIV-treatment amount of the pharmaceutically acceptable salt of β-isomerof a substituted 2'-deoxy-5-fluoro-3'-thiacytidine having the formula:##STR14## wherein Y is independently selected from the group consistingof H, alkyl, substituted alkyl, cycloalkyl and acyl; andR is selectedfrom the group consisting of H, hydroxy, oxyacyl, monophosphate,diphosphate, and triphosphate.
 12. A method for treating a human havingan HIV infection comprising administering to the human an effectiveHIV-treatment amount of a pharmaceutically acceptable salt of a compoundof the formula: ##STR15## wherein Y is monophosphate, diphosphate, ortriphosphate.
 13. A method for treating a human having an HIV infectioncomprising administering to the human an effective HIV treatment amountof the pharmaceutically acceptable salt of a compound of the formula:##STR16## wherein Y is independently selected from the group consistingof H, alkyl, cycloalkyl and acyl; andR is selected from the groupconsisting of H, hydroxyl, oxyacyl, monophosphate, diphosphate, andtriphosphate.
 14. The method of claim 1, wherein the compound isadministered in a pharmaceutically acceptable carrier.
 15. The method ofclaim 4, wherein the compound is administered in a pharmaceuticallyacceptable carrier.
 16. The method of claim 8, wherein the compound isadministered in a pharmaceutically acceptable carrier.
 17. The method ofclaim 9, wherein the compound is administered in a pharmaceuticallyacceptable carrier.
 18. The method of claim 10, wherein the compound isadministered in a pharmaceutically acceptable carrier.
 19. The method ofclaim 11, wherein the compound is administered in a pharmaceuticallyacceptable carrier.
 20. The method of claim 12, wherein the compound isadministered in a pharmaceutically acceptable carrier.
 21. The method ofclaim 13, wherein the compound is administered in a pharmaceuticallyacceptable carrier.